Structures of Yeast Apa2 Reveal Catalytic Insights into a Canonical Ap₄A Phosphorylase of the Histidine Triad Superfamily

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• 作者：Wen-Tao Hou ; Wen-Zhe Li ; Yuxing Chen ; Yong-Liang Jiang ; Cong-Zhao Zhou
• 关键词：Ap4A ; 5&prime ; 5&Prime ; &prime ; -P1 ; P4-tetraphosphate ; Ap3A ; 5&prime ; 5&Prime ; &prime ; -P1 ; P3-tetraphosphate ; Ap5A ; 5&prime ; 5&Prime ; &prime ; -P1 ; P5-tetraphosphate ; GalT ; galactose-1-phosphate uridylyltransferase ; HIT ; histidine triad ; HPLC ; high-performance li
• 刊名：Journal of Molecular Biology
• 出版年：2013
• 出版时间：9 August, 2013
• 年：2013
• 卷：425
• 期：15
• 页码：2687-2698
• 全文大小：2642 K

文摘

The homeostasis of intracellular diadenosine 5¡ä,5¡å¡ä-P¹,P⁴-tetraphosphate (Ap₄A) in the yeast Saccharomyces cerevisiae is maintained by two 60 % sequence-identical paralogs of Ap₄A phosphorylases (Apa1 and Apa2). Enzymatic assays show that, compared to Apa1, Apa2 has a relatively higher phosphorylase activity towards Ap₃A (5¡ä,5¡å¡ä-P¹,P³-tetraphosphate), Ap₄A, and Ap₅A (5¡ä,5¡å¡ä-P¹,P⁵-tetraphosphate), and Ap₄A is the favorable substrate for both enzymes. To decipher the catalytic insights, we determined the crystal structures of Apa2 in the apo-, AMP-, and Ap₄A-complexed forms at 2.30, 2.80, and 2.70 ? resolution, respectively. Apa2 is an ¦Á/¦Â protein with a core domain of a twisted eight-stranded antiparallel ¦Â-sheet flanked by several ¦Á-helices, similar to the galactose-1-phosphate uridylyltransferase (GalT) members of the histidine triad (HIT) superfamily. However, a unique auxiliary domain enables an individual Apa2 monomer to possess an intact substrate-binding cleft, which is distinct from previously reported dimeric GalT proteins. This cleft is perfectly complementary to the favorable substrate Ap₄A, the AMP and ATP moieties of which are perpendicular to each other, leaving the ¦Á-phosphate group exposed at the sharp turn against the catalytic residue His161. Structural comparisons combined with site-directed mutagenesis and activity assays enable us to define the key residues for catalysis. Furthermore, multiple-sequence alignment reveals that Apa2 and homologs represent canonical Ap₄A phosphorylases, which could be grouped as a unique branch in the GalT family.